In response to NIAID's Strategic Plan for Biodefense Research, we have initiated a program to discover and develop broad-spectrum antimicrobials that target multiple bacterial pathogens through inhibition of alanine racemase, an enzyme in the D-alanine metabolic pathway that is essential for bacterial cell wall synthesis. This pathway is highly conserved in bacteria, virtually absent in humans, and is a validated target of cycloserine, a commercially available antibiotic that is limited by off-target toxicity. The long-term goal of this project is to develop safe and effective small molecule therapeutics that target alanine racemase in a variety of bacterial pathogens including those of biodefense importance. A proprietary high-throughput screening assay using recombinant Mycobacterium tuberculosis alanine racemase was developed to screen for novel enzyme inhibitors. A pilot screen identified promising 'hits' with antibacterial activity against M. tuberculosis, the bacterium that causes tuberculosis. At least one of these hits inhibits clinical isolates that are multi-drug resistant (MDRTB). This project will broaden the number and utility of our alanine racemase inhibitors as anti-infectives effective against multiple NIAID bacterial priority pathogens. Starting with hit compounds active against MDRTB, iterative medicinal chemistry and screening will be used to broaden the spectrum of activity of these agents against Bacillus anthracis, the causative agent of anthrax, and methicillin-resistant Staphylococcus aureus (MRSA), the causative agent of life threatening bacteremia. This project will be performed by a multidisciplinary team of investigators from academia and industry with expertise encompassing such areas as high-throughput screening, small molecule medicinal chemistry, and structural biology who share a vision of a novel broad-spectrum alanine racemase inhibitor as a therapeutic against multiple bacterial pathogens. This project will employ methods of high-throughput screening and medicinal chemistry coupled with structural biology and animal models of MDR-TB, MRSA and anthrax to advance screening hits towards therapeutic leads. RELEVANCE (See instructions): The long-term product goal of this project is a broad-spectrum therapeutic to treat bacterial infections of public health and biodefense importance. Accomplishing this goal would enhance our national preparedness for natural, accidental and intentional exposure to several pathogens, and help address emerging microbial drug resistance.